Tumor-Associated-Macrophage-Membrane-Coated Nanoparticles for Improved Photodynamic Immunotherapy.

Cell-membrane-coated nanoparticles have emerged as a promising antitumor therapeutic strategy. However, the immunologic mechanism remains elusive, and there are still crucial issues to be addressed including tumor-homing capacity, immune incompatibility, and immunogenicity. Here, we reported a tumor-associated macrophage membrane (TAMM) derived from the primary tumor with unique antigen-homing affinity capacity and immune compatibility. TAMM could deplete the CSF1 secreted by tumor cells in the tumor microenvironment (TME), blocking the interaction between TAM and cancer cells. Especially, after coating TAMM to upconversion nanoparticle with conjugated photosensitizer (NPR@TAMM), NPR@TAMM-mediated photodynamic immunotherapy switched the activation of macrophages from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state, induced immunogenic cell death, and consequently enhanced the antitumor immunity efficiency via activation of antigen-presenting cells to stimulate the production of tumor-specific effector T cells in metastatic tumors. This TAM-membrane-based photodynamic immunotherapy approach offers a new strategy for personalized tumor therapy.

Combining Gemcitabine-Loaded Macrophage-like Nanoparticles and Erlotinib for Pancreatic Cancer Therapy.

Pancreatic cancer is a lethal malignancy with a dismal prognosis. Gemcitabine is currently used to treat pancreatic cancer, but it is limited by significant toxicity. Clinical trials on the combination of gemcitabine and erlotinib reported unsatisfactory outcomes along with concerns of toxicity. The encapsulation of chemotherapy drugs in polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) can alleviate toxicity through targeted delivery and sustained release. In addition, camouflaging the NPs with a macrophage membrane can evade the immune system and further improve tumor homing. We designed gemcitabine-loaded PLGA NPs with a macrophage membrane coating (MPGNPs) to reduce drug toxicity and increase the accumulation in the tumor. The combination of MPGNPs and erlotinib synergistically inhibited pancreatic cancer cell proliferation in vitro and in vivo by targeting the PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling pathways. The MPGNPs were also able to evade phagocytosis and achieve passive targeting to the pancreatic tumors. The combination of MPGNPs and erlotinib showed synergistic anti-tumor efficacy in vitro and in vivo. This study provides a proof-of-concept for treating pancreatic cancer with a combination of MPGNPs and erlotinib.

Unexpected diradical character and large magnetic spin coupling in modified porphyrins induced by inverting pyrrole rings.

Porphyrin derivatives with inverted pyrrole rings have been experimentally synthesized, and their relevant electronic and magnetic properties have great application prospects in terms of electronic devices. In this work, we rationally design the structures and computationally investigate the electronic properties of porphine and Mg/Zn-porphyrin derivatives with two inverted pyrrole rings, i.e. the dipyrrole-inverted porphine and Mg/Zn-porphyrin analogues (1NN-2H, 2NN-2H, 1NN-Mg, 2NN-Mg, 1NN-Zn and 2NN-Zn), at the B3LYP/6-311G(d,p) level. The main structural characters of these porphyrin derivatives are that the [double bond splayed left]NH units of two pyrrole rings are inverted outwards and the porphyrin-like macrocycles are distorted from square to diamond shapes. More interestingly, these dipyrrole-inverted porphyrin derivatives present diradical characters with noticeably large antiferromagnetic spin coupling constants, i.e.-982.2/-936.3 cm-1 for 1NN-2H/2NN-2H, -796.3/-764.2 cm-1 for 1NN-Mg/2NN-Mg and -1044.5/-1055.2 cm-1 for 1NN-Zn/2NN-Zn, but their monopyrrole-inverted counterparts do not. Examinations of the orbital properties featuring large occupation numbers of the lowest unoccupied natural orbitals and two singly occupied molecular orbitals that are polarized in opposite directions also confirm these findings. These porphyrin derivatives have small singlet-triplet energy gaps and small energy gaps between the highest occupied molecular orbital and lowest unoccupied molecular orbital of the closed-shell singlet states. These are conducive to the emergence of diradical character and large spin coupling constants. Furthermore, the spin-alternation analyses show that each dipyrrole-inverted porphyrin derivative has two resonant structures featuring two spin-opposite single electrons, in agreement with the observed antiferromagnetic spin couplings. This work provides novel insights into the electronic structures and properties of the porphyrin derivatives with modified structures and also provides helpful information for the rational design, synthesis and characterization of new porphyrin-based magnets.

MiR-155 targets PTCH1 to mediate endothelial progenitor cell dysfunction caused by high glucose.

Endothelial progenitor cells (EPCs) are involved in diabetes-associated complications, including diabetic foot ulcer (DFU). Recent reports showed that miR-155 downregulation promotes wound healing in diabetic rats and ameliorates endothelial injury induced by high glucose, but its role in DFU is unknown. We found that miR-155 was overexpressed in EPCs from patients with DFU and in high glucose-induced EPCs from healthy people. Reductions in cell viability, migration, tube formation and nitric oxide production, as well as increases in lactated hydrogenase, cell apoptosis, and reactive oxygen species induced by high glucose, were enhanced by miR-155 overexpression and restrained by miR-155 inhibition. Additionally, dual-luciferase reporter assay demonstrated that miR-155 directly targeted the 3' untranslated region of patched-1 (PTCH1), a receptor of the sonic hedgehog signaling pathway, and downregulated the mRNA and protein expression of PTCH1. qRT-PCR and Western blot results revealed that the PTCH1 was downregulated in EPCs treated with high glucose. Silencing PTCH1 by PTCH1 siRNA alleviated the protective effect of anti-miR-155 on high glucose-induced EPC dysfunction. Our results indicate that miR-155 worsened high glucose-induced EPC function by downregulating PTCH1. These findings suggest that miR-155 may be a potential therapeutic target for DFU.

Spin coupling interactions in C[double bond, length as m-dash]C or B-B-cored porphyrin-mimetic graphene patch nitroxide diradicals.

In view of the unique structures and promising applications of porphyrins and their derivatives, exploration of their various properties has continued to a hot topic. In this work, we combine porphyrin-mimetic graphene patches which are core-modified by a C[double bond, length as m-dash]C or a B-B unit and two nitroxide radical groups to construct a series of novel diradical molecules (the CC-cored or BB-cored molecules). The spin coupling constants (J) of diradicals were calculated at the (U)B3LYP/6-311G(d,p) level by considering the different linking modes of two nitroxide groups. The results indicate that different core modification considerably affects the J values of such diradicals, and the linking modes can tune the sizes and signs of J, changing their magnetic coupling interactions with different magnitudes and the signs of J from antiferromagnetic to ferromagnetic or vice versa. More interestingly and importantly, the spin coupling interactions of the CC-cored molecules can also be tuned by stretching the core unit C-C bond, suggesting the possibility of activating specific vibrational modes of the CC-cored diradicals by energy pulses to yield variable J coupling magnitudes. On the other hand, for the BB-cored molecules, two-electron reduction can switch or tune their magnetism from ferromagnetic to antiferromagnetic. The essence of all observations is further analyzed from the structural effects and orbital and spin density distributions. The findings about magnetic regulation in these core-modified porphyrin-mimetic graphene patch nitroxide diradicals further expand the field of molecular magnets and provide a rational theoretical basis for designing novel building blocks of magnetic functional molecular materials.

Tuning the Spin Coupling Interactions in the Nitroxide-Based Bisphenol-Like Diradicals.

The intramolecular spin coupling interactions of bisphenol-like trinary-bridged diradicals [nitroxide-(para/meta)phenylene-X-phenylene(para/meta)-nitroxide, X=C=CH2 , O, BH, NH and SO2 ] were explored with an emphasis on the tuning role of the X coupler at the (U)B3LYP/6-311++G(d,p) level. Our results indicate that all designed trinary-bridged diradicals featuring a V-type structure with a bending angle of 104-130° and torsional angles of two phenylene rings being 20-90° exhibit different diradical character and magnetism, depending on the structures and properties of the X bridges. More interestingly, although meta/para-phenylene supports a ferromagnetic (FM)/antiferromagnetic (AFM) coupling, their combinations by using X as a trinary bridge can mediate spin coupling, but the coupling magnitude strongly depends on X. In general, a para/para or meta/meta combination with X leads to an open-shell singlet ground state and thus AFM, but the meta/meta combination considerably decreases the spin coupling interaction. In contrast, a para/meta combination with X produces a triplet ground state and FM. Their combination with a single-electron conjugation end coupler (C=CH2 ) leads to an inverse coupling regularity. All results can be reasonably explained by the spin alternation rule, molecular structures, and properties. In particular, detailed spin coupling mechanisms are suggested to involve cooperative through-space and through-bond pathways with different levels of cooperativity. This is rationalized with the X-induced bending of the diradicals not only modifying the through-bond (extended π conjugation) pathway but also provididng a through-space (face-to-face vs. side-to-side π-π interaction) possibility for spin coupling, in conjunction with twisting of the phenylene rings. Different contributions of the through-space and through-bond couplings are quantitatively distinguished and depend on the structure and property of the X coupler. Clearly, this work reports interesting aspects of the trinary bridged diradicals and also provides important information for the design of molecules for functional magnetic materials and tuning their magnetic properties.

Hsa_circ_0001326 inhibited the proliferation, migration, and invasion of trophoblast cells via miR-145-5p/TGFB2 axis.

PROBLEM: Preeclampsia (PE) is an obstetric disease involving multiple systems, which account for maternal and fetal complications and increased mortality. Circular RNAs (circRNAs) were recently deemed to associate with the pathogenesis of PE. This study aims to clarify the correlation between circRNA hsa_circ_0001326 and PE and explore its biological function in PE. METHOD OF STUDY: The expression of hsa_circ_0001326 in PE placentas was detected by real-time quantitative PCR (qRT-PCR). After overexpressing or inhibiting hsa_circ_0001326 in trophoblast cells, the cell growth, migration, and invasion were evaluated by Cell Counting Kit-8 (CCK-8) and transwell assays. Western blot assay was applied to detect the epithelial-mesenchymal transition (EMT) proteins, E-cadherin and Vimentin. Furthermore, a dual-luciferase reporter assay was applied to verify the binding sites of hsa_circ_0001326, miR-145-5p, and transforming growth factor beta 2 (TGFB2). RESULTS: Hsa_circ_0001326 was found to be higher expressed in PE placentas than in normal placentas. Furthermore, hsa_circ_0001326 played a negative regulating role in trophoblast cell viability, migration, and invasion. Overexpression of hsa_circ_0001326 inhibited the viability, migration, and invasion of trophoblast cells, while inhibition of hsa_circ_0001326 showed opposite effects. Mechanistically, hsa_circ_0001326 sponged miR-145-5p to elevate TGFB2 expression in trophoblast cells. CONCLUSION: This study provided evidence that the up-regulated hsa_circ_0001326 in PE restrained trophoblast cells proliferation, migration, and invasion by sponging miR-145-5p to elevate TGFB2 expression. Our results might provide a novel insight into the role of hsa_circ_0001326 in the pathogenesis of PE.

A Bimetallic Metal-Organic-Framework-Based Biomimetic Nanoplatform Enhances Anti-Leukemia Immunity via Synchronizing DNA Demethylation and RNA Hypermethylation.

Epigenetic-alterations-mediated antigenicity reducing in leukemic blasts (LBs) is one of the critical mechanisms of immune escape and resistance to T-cell-based immunotherapy. Herein, a bimetallic metal-organic framework (MOF)-based biomimetic nanoplatform (termed as AFMMB) that consists of a DNA hypomethylating agent, a leukemia stem cell (LSC) membrane, and pro-autophagic peptide is fabricated. These AFMMB particles selectively target not only LBs but also LSCs due to the homing effect and immune compatibility of the LSC membrane, and induce autophagy by binding to the Golgi-apparatus-associated protein. The autophagy-triggered dissolution of AFMMB releases active components, resulting in the restoration of the stimulator of interferon genes pathway by inhibiting DNA methylation, upregulation of major histocompatibility complex class-I molecules, and induction of RNA-methylation-mediated decay of programmed cell death protein ligand transcripts. These dual epigenetic changes eventually enhance T-cell-mediated immune response due to increased antigenicity of leukemic cells. AFMMB also can suppress growth and metastases of solid tumor, which was suggestive of a pan-cancer effect. These findings demonstrate that AFMMB may serve as a promising new nanoplatform for dual epigenetic therapy against cancer and warrants clinical validation.

Up-regulation of caveolin 1 mediated by chitosan activates Wnt/ ß-catenin pathway in chronic refractory wound diabetic rat model.

Diabetes mellitus (DM) can be implicated in the perturbations of vascular integrity and the dysfunction of angiogenesis. Chitosan has the advantage of promoting the vascular endothelial cell proliferation. However, the molecular mechanism of action in the promotion of wound healing by chitosan derivatives is still debated. In the current study, DM with chronic wound (CW) model rats were prepared and treated with chitosan. Vascular endothelial cells isolated from granulation tissues were conducted by RNA sequencing. Two thousand three hundred and sixteen genes were up-regulated, while 1,864 genes were down-regulated after chitosan treatment compared to CW group. Here, we observed that caveolin 1 (CAV1) was highly expressed induced by chitosan. Furthermore, we observed that CAV1 knockdown could compromise the activation of Wnt pathway by reduction of ß-catenin in rat aortic endothelial cells (RAOECs) and brain endothelium four cells (RBE4s). Moreover, we determined a direct interaction between CAV1 and ß-catenin by IP assay. The C-terminus of CAV1 and ß-catenin (24 to 586 amino acids) contributed to the interaction of these two proteins. Finally, the protein docking analysis indicated that the fragments of ß-catenin (253-261 'FYAITTLHN' and 292-303 'KFLAITTDCLQI') might have affected the structure by CAV1 and facilitated the resistance to degradation. Taken together, our study demonstrates that chitosan can up-regulate CAV1 expression, and CAV1 can interact with ß-catenin for promotion of canonical Wnt signaling pathway activity. Our results deepens the molecular mechanism of the Wnt pathway in vascular endothelial cells and is beneficial to developing new targets to assist in enhancing the pharmacological effect of chitosan on wound healing and angiogenesis against DM.

Analysis of Distributions of HPV Infection in Females with Cervical Lesions in the Western District of Beijing Chaoyang Hospital.

Objective: To analyze the distribution of human papilloma virus (HPV) infection in women with cervical lesions of different grades and analyze the relationship of high-risk HPV and cervical lesions in order to facilitate targeted prevention. Methods: The infection status of HPV subtype was statistically analyzed in patients who underwent colposcopy examination from April 2017 to June 2019. Results: The infection rate of HPV was 81.4% in chronic cervicitis, 82.9% in 1ow-grade squamous intraepithelial lesion (LSIL), 63.7% in HSIL (high-grade squamous intraepithelial lesion), and 50% in cervical squamous cell carcinoma (CSCC). Among the 16 high-risk HPV types, the top six HPV types with the comprehensive infection rates were HPV16 > HPV52 > HPV58 > HPV18 > HPV51 > HPV53 in turn, and the infection rates were 23.3%, 14.8%, 13.3%, 9.8%, 9.2%, and 8.8%, respectively. The infection rates of HPV16 in chronic cervicitis group, LSIL group, and HSIL group were significantly different. There was no significant difference in the injection rates of HPV52, HPV58, and HPV18 among the three groups. HPV infection rates were highest in the 31-40 years old group, followed by the 41-50 years old group. Conclusion: The distribution of different types of HPV varies in different tissue types, which can be used to develop relevant vaccines to achieve better prevention and treatment of cervical cancer.

Fluorescent nanorods based on 9,10-distyrylanthracene (DSA) derivatives for efficient and long-term bioimaging.

Fluorescent nanoparticles based on 9,10-distyrylanthracene (DSA) derivatives (4,4'-((1E,1'E)-anthracene-9,10-diylbis(ethene-2,1-diyl))bis(N,N-dimethylaniline) (NDSA) and 4,4'-((1E,1'E)-anthracene-9,10-diylbis(ethene-2,1-diyl))dibenzonitrile (CNDSA)) were prepared using an ultrasound aided nanoprecipitation method. The morphologies of the fluorescent nanoparticles could be controlled by adjusting the external ultrasonication time. NDSA or CNDSA could form spherical nanodots (NDSA NDs, CNDSA NDs) in a THF-H2O mixture with an 80% or 70% water fraction when the ultrasonication time was 30 s. When the ultrasonication time was prolonged to 10 min, NDSA and CNDSA could assemble into nanorods (NDSA NRs, CNDSA NRs). Meanwhile, the sizes of NDSA NRs and CNDSA NRs could be controlled by adjusting the water content in the mixture. As the water fraction was increased from 60% to 80%, the sizes of NDSA and CNDSA nanorods or nanodots reduced from 238.4 nm to 140.3 nm, and 482 nm to 198.4 nm, respectively. When the water fraction was up to 90%, irregular morphologies of NDSA and CNDSA could be observed. The nanoparticles exhibited intense fluorescence emission, good anti-photobleaching properties, as well as excellent stability and biocompatibility. In vitro cell imaging experiments indicated that the nanorods prepared by this simple method had the potential to be used for efficient and noninvasive long-term bioimaging.

TICT-Based Near-Infrared Ratiometric Organic Fluorescent Thermometer for Intracellular Temperature Sensing.

Fluorescent thermometers with near-infrared (NIR) emission play an important role in visualizing the intracellular temperature with high resolution and investigating the cellular functions and biochemical activities. Herein, we designed and synthesized a donor-Π-acceptor luminogen, 2-([1,1'-biphenyl]-4-yl)-3-(4-((E)-4-(diphenylamino)styryl) phenyl) fumaronitrile (TBB) by Suzuki coupling reaction. TBB exhibited twisted intramolecular charge transfer-based NIR emission, aggregation-induced emission, and temperature-sensitive emission features. A ratiometric fluorescent thermometer was constructed by encapsulating thermosensitive NIR fluorophore TBB and Rhodamine 110 dye into an amphiphilic polymer matrix F127 to form TBB&R110@F127 nanoparticles (TRF NPs). TRF NPs showed a good temperature sensitivity of 2.37%·°C-1, wide temperature response ranges from 25 to 65 °C, and excellent temperature-sensitive emission reversibility. Intracellular thermometry experiments indicated that TRF NPs could monitor the cellular temperature change from 25 to 53 °C for Hep-G2 cells under the photothermal therapy agent heating process, indicating the considerable potential applications of TRF NPs in the biological thermometry field.

Bortezomib-Encapsulated CuS/Carbon Dot Nanocomposites for Enhanced Photothermal Therapy via Stabilization of Polyubiquitinated Substrates in the Proteasomal Degradation Pathway.

Photothermal therapy (PTT) is an emerging therapeutic strategy in the treatment of cancer; however, a critical challenge remains in the rational design of synergistic nanoparticles as a potential photothermal transduction agent that can effectively enhance the therapeutic outcome of PTT for tumor ablation. Herein, we rationally designed, developed, and characterized hollow-structured CuS nanoparticles composited with carbon dots (CuSCDs), which demonstrated excellent photothermal conversion efficiency under a 808 nm laser irradiation with enhanced biocompatibility and reduced toxicity. Following coating with a macrophage membrane hybridized with T7 peptide on the surface of the proteasome inhibitor loaded CuSCD, CuSCDB@MMT7 exhibited targeted specificity to cancer cells with the characteristics of immunity escaping and enhanced transferrin receptor-mediated endocytosis. Predominantly, CuSCDB@MMT7-triggered PTT exhibited the accumulation of the polyubiquitinated tumor suppressor protein that is heat stabilized under NIR induced hyperthermia, facilitating augmented tumor cell apoptosis and the attenuated metastasis. This study provides a proof-of-concept for the proteasome inhibitor-loaded CuS/carbon dot nanocomposite-PTT strategy and highlights a promising therapeutic strategy for realizing enhanced therapeutic outcomes for effective clinical cancer therapy.

Hybrid membrane camouflaged copper sulfide nanoparticles for photothermal-chemotherapy of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Biomimetic nanoparticles (NPs) coated with cell membranes show enhanced biocompatibility and specificity for homotypic cells, and have gained considerable attention for targeted anti-tumor therapy. We constructed cancer cell-macrophage hybrid membrane-coated near infrared (NIR)-responsive hollow copper sulfide nanoparticles encapsulating sorafenib and surface modified with anti-VEGFR (CuS-SF@CMV NPs). These CuS-SF@CMV NPs expressed the characteristic membrane proteins of both cancer cells and macrophages, and selectively accumulated in cancer cells in vitro and tumors in vivo, compared to the CuS NPs. In addition, the CuS-SF@CMV NPs achieved synergistic photo-thermal and chemotherapy in cancer cells upon NIR irradiation, with 94.3% inhibition of tumor growth in a murine hepatoma model. While the initial increase in temperature rapidly killed the tumor cells, sorafenib and the anti-VEGFR antibody sustained the tumor killing effect by respectively inhibiting tumor cell proliferation and angiogenesis via the Ras/Raf/MEK/ERK and PI3K/AKT pathways. Taken together, the CuS-SF@CMV NPs have immune evasion, tumor cell targeting and drug loading capacities, along with an inherent photo-thermal conversion ability, making them ideal for synergistic photo-thermal/chemo therapy against HCC. STATEMENT OF SIGNIFICANCE: We created cancer cell-macrophage hybrid membrane-coated hollow CuS NPs encapsulating sorafenib and surface modified with anti-VEGFR antibodies (CuS-SF@CMV). These CuS-SF@CMV NPs enhanced synergistic PTT and chemotherapy against hepatoma cells through homotypic cell targeting, immune escape and inhibition of a tumorigenic signaling pathway. A long-term inhibition of tumor growth and metastasis was achieved owing to the rapid destruction of the cancer cells through photo-thermal conversion by the CuS NPs, and sustained clearance of the tumor cells by sorafenib and anti-VEGFR antibodies. Our findings suggest that CuS-SF@CMV NPs present great treating effects in preclinical models of HCC, providing the framework for further study in clinical trials to improve patient outcome in hepatocellular carcinoma.

Multifunctional Gold Nanoparticles Overcome MicroRNA Regulatory Network Mediated-Multidrug Resistant Leukemia.

Resistance to chemotherapy and molecularly targeted therapies is a major problem in current leukemia treatments. Here, we investigated cross-talk between the miR-221 network and P-glycoprotein (P-gp) in doxorubicin-induced drug resistance of leukemia cells. Multifunctional gold nanoparticles were designed and synthesized to co-deliver three anticancer agents, AS1411, doxorubicin and anti-221, for improving leukemia treatment efficacy. These nanoparticles significantly inhibited the proliferation and clonogenic potential, and induced apoptosis of drug-resistant leukemia cells. The decreased growth of drug-resistant cells induced by these nanoparticles was associated with marked downregulation of miR-221 and DNMT1, leading to restored p27kip1 and p15ink4b tumor suppressor expression, as well as miR-221-mediated reduction of P-gp expression. Finally, primary blasts derived from leukemia patients experiencing chemoresistant relapse that were exposed to these nanoparticles were sensitized to doxorubicin, as evidenced by suppression of leukemic cell growth and a significant reduction of the doxorubicin IC50 value. Our findings provide proof of concept that this novel drug delivery system can precisely reverse the multidrug resistant leukemia phenotype based on preclinical models of leukemia, providing the framework for future clinical trials aimed at overcoming drug resistance and improving patient outcome.

Phenethyl isothiocyanate suppresses receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis by blocking activation of ERK1/2 and p38 MAPK in RAW264.7 macrophages.

Osteoclastogenesis is induced by differentiation of hemopoietic cells of monocyte-macrophage lineage into bone-resorbing osteoclasts. The process is initiated by receptor activator of NF-kappaB ligand (RANKL) and resultant activation of mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK)1/2, as well as the NFkappaB pathway. Phenethyl isothiocyanate (PEITC), a phytochemical present in various cruciferous plants, has been shown to disrupt those signaling pathways in several cell types. In this study, we examined the efficacy of PEITC for suppressing RANKL-induced osteoclastogenesis in RAW264.7 murine macrophages and addressed the underlying molecular mechanisms. PEITC (2-10 microM) suppressed osteoclastogenesis in a concentration dependent manner, as detected by tartarate-resistant acid phosphatase (TRAP) activity and microscopic observations. RANKL-up-regulated extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) activities were attenuated by PEITC, whereas c-Jun N-terminal kinase (JNK1/2) activation was increased. PEITC also abrogated the RANKL-induced degradation of IkappaB-alpha, a suppressive partner of nuclear factor kappaB (NFkappaB), thereby inhibiting transcription activity, as detected by a reporter assay. In addition, PEITC reduced the level of NFkappaB-dependent mRNA expression of nuclear factor of activated T cell (NFAT)c1, a master regulator of osteoclastogenesis. Our results indicate that PEITC is a promising agent for treatment of osteoclastogenesis with a reasonable action mechanism.

Citrus nobiletin suppresses bone loss in ovariectomized ddY mice and collagen-induced arthritis in DBA/1J mice: possible involvement of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis regulation.

Bone resorption is known to accelerate during the onset of several disorders, including osteoporosis (OP) and rheumatoid arthritis (RA). Some epidemiological surveys have suggested that a high intake of vegetables and fruits has an inverse relation to such disease incidence, though the number of active constituents elucidated thus far is limited. In the present study, we examined the efficacy of various food phytochemicals using two animal models. First, female ddY mice were ovariectomized (OVX) or sham-operated (sham), after which five different compounds (phenethyl isothiocyanate, zerumbone, auraptene, 1'-acetoxychavicol acetate, and nobiletin) were administered separately to OVX mice with a mini-osmotic pump at doses of 0.25 or 0.5 mg/day for 4 weeks, with 17beta-estradiol (E_{2}, 0.03 microg/day) used as a positive control. Nobiletin, in contrast to the other tested phytochemicals, significantly (P<0.05) suppressed the reduction of whole bone mineral density by 61%, which was comparable to or higher than the efficacy of E_{2}. Next, nobiletin given as an i.p. administration at 20 mg/kg of body weight, but not 2 mg/kg, to male DBA/1J mice every 2 days for 12 days led to a marked decrease in type II collagen-induced arthritis by 45% (P < 0.05). Furthermore, the flavonoid (4-50 microM) attenuated receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis of RAW264.7 cells, as detected by tartarate-resistant acid phosphatase activity and microscopic observations. Of note, nobiletin also suppressed RANKL-activated extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2, and p38 mitogen-activated protein kinase activities, and thereby regulated the promoter activation of nuclear factor kappaB (NFkappaB) and activator protein-1, key transcription factors for differentiation. Together, our results suggest that nobiletin is a promising phytochemical for the prevention or treatment of osteoclastogenesis-related disorders, including OP and RA, with reasonable action mechanisms.